Fluorescent and chemiluminescent signal probes have recently come into widespread use, in place of conventional radioactive signal probes for the detection and quantification of nucleic acids, sequence analysis, hybridization assays, immunological measurements, and so forth. In general, the optical detection method is safer and simpler than detection methods involving the use of radiation, and its spectroscopic devices have be advanced in recent years, for good selectivity and sensitivity comparable to that of radiochemical methods.
However, there remains a great need for higher sensitivity of detection. For instance, if molecule-countable level of DNA or RNA could ultimately be detected, it would be an easy matter to analyze the genomic DNA or RNA in a single cell. Also, there has been a need for the development of an excellent signal probe and its technology for easily and rapidly detecting differences in the molecular level of nucleic acids between normal and abnormal cells and in the cells of tissues or organs.
With efforts to increase the sensitivity of detection, various studies have being conducted on the research points of labeling reagents, signal probe and detector device.
The genomic DNA in a single cell can be detected by conventional fluorescent-labeling or dye-staining technique, when the nucleotides of DNA are polymerized in kilo-units or larger. However, it is extremely difficult to detect a difference of a single base in a DNA sequence (SNPS: Single Nucleotide Polymorphisms) or a difference of a few bases in a DNA sequence, even with today's fluorescent labeling technique. Thus, a sample (such as DNA) quantity at least 10 fmol is required even when using laser fluorescent dyes that are known as highly sensitive fluorescent dyes.
Also, when a fluorescent dye is used for detection, even with multiple fluorescent dyes for intensifying, quenching phenomenon may occur according to the increase of the fluorescent dyes, which may lead to the unsatisfactory detection sensitivity. Accordingly, it is no easy to detect an extremely small mutation points in a DNA sequence with fluorescent dyes.
There have also been proposed fluorescent and chemiluminescent detection methods that make use of an enzyme probe operating on the principle of amplifying the sensitivity by enzyme reaction time (Stephan Beck and Hubert Koster, Anal. Chem., 60, 2258-2270 (1990)). However, the degree of sensitivity amplification in their methods is dependent on the enzyme reaction time. If there is only a very small amount of target substance, therefore the enzyme reaction takes a long time in order to raise the sensitivity, it is difficult to obtain data rapidly, and the background noise is also amplified at the same time, which can result in inferior resolution for the detection.
In an attempt to achieve higher detection sensitivity, various luminescent detection methods have been proposed, in which a polymer comprising many fluorescent or chemiluminescent substances is used as a labeling agent. Specific examples include a method in which an acridinium compound having a plurality of acridinium rings, or a complex thereof, is used as a chemiluminescent labeling agent (Japanese Laid-Open Patent Applications H6-158039 and H9-100415); a method in which an acridan group-containing polymer having a repeating domain of acridan group joined portion [a (meth)acrylic acid (co)polymer, polyvinyl alcohol, or polyethylene glycol (meth)acrylate copolymer] is used as a chemiluminescent labeling agent (Japanese Laid-Open Patent Applications H7-330838, H8-113611, and H9-302033); and a method in which an organic polymer (such as a synthetic peptide or polyvinylphenol) bound with a plurality of luminescent substance such as luminol or lucigenin is used as a chemiluminescent labeling agent in immunoassay (Japanese Laid-Open Patent Applications S58-61468, S58-137759, and S58-137760). Also, although not aimed at highly sensitive detection, Japanese Laid-Open Patent Application H2-102203 reports that the binding of a luminescent substance such as luminol to a polyvinyl alcohol in order to increase the water solubility of the luminescent substance, and this luminescent substance is usable as a reagent for evaluating the bio-membrane permeability. However, as shown from the above-mentioned publications, there is no report that is disclosed a compound (including a polymer) comprising one or more fluorescent or chemiluminescent substances bound thereto, and also being capable of amplifying luminescent intensity by themselves mutually forming complexes sequentially, and a technique for amplification of the luminescent intensity using the said compound.